The primary goal is to increase understanding of the initial molecular targets for ethanol ligand-gated ion channels (LGICs). The proposal builds on our previous work which found that increased atmospheric pressure (pressure) antagonizes ethanol at the behavioral, biochemical and molecular levels and indicates that pressure can provide unique insights into the molecular structures on which ethanol acts in LGICs. The proposed work will: 1) Test hypotheses regarding the sites of action of ethanol suggested by our recent findings in glycine receptors (GlyRs) using standard molecular approaches including cysteine mutagenesis and sulfhydryl-specific labeling and 2) Use the unique qualities of pressure antagonism to test hypotheses regarding the sites of action for ethanol in GlyR and other LGICs that are difficult to address with molecular techniques alone. This proposal focuses on GlyR and GABAAR expressed in oocytes using two-electrode voltage clamp and will begin to investigate other LGICs. Confirmatory studies of mutant receptor properties will be conducted in HEK 293 and/or NG 108 cells using whole cell and patch clamp techniques. Aim 1 - Use pressure and molecular manipulations to identify and test novel sites of action for ethanol in GlyRs and GABAARs. Aim 2 - Use pressure to screen for possible structural and/or functional similarities between sites of action for ethanol across LGICs within and outside of the nicotinic cholinergic (Cys-loop) superfamily of LGICs. The proposed work builds on differences we found in sensitivity to pressure antagonism of ethanol between alpha1GlyR (pressure antagonism sensitive) and alpha2GlyR (pressure antagonism insensitive). The findings of Aims I and 2 will be used to modify existing, or to construct new, molecular models of ethanol's sites of action in LGICs. Overall, this work will contribute to our long-term goal, which is to identify specific targets at which therapeutically relevant agents can be directed to reduce the social problems, loss of lives and tremendous economic costs resulting from the misuse and abuse of alcohol.